Embryonic stem cell (ESC) differentiation is a potentially powerful approach for generating a renewable source of cells for regenerative medicine. During embryonic development, the inner cell mass (ICM) from which ESCs are derived, differentiate into specific lineages based on a series of temporally and spatially regulated signals. However, the directed differentiation of ESCs in tissue culture typically lacks such tight control and homogenous environments. In most protocols, cell aggregate intermediates called embryoid bodies (EBs) are generated. EBs recapitulate many features of ICM cells during embryonic development and give rise to a wide spectrum of cell types. However, the resulting EBs are typically heterogeneous in size and shape and lack proper temporal and spatial signaling. These variations could be a potential source of heterogeneity in differentiation and prevent uniform and directed cellular differentiation. The goal of this project is 1) to develop and characterize a microfabricated platform to control the homogeneity, size and shape of EBs and 2) to analyze the spatial distribution and frequency of differentiation of definitive and primitive (visceral) endoderm cells as a function of EB size in suspension cultures and within the microfabricated platform. In this project we will develop methods of controlling the homogeneity and differentiation of mouse embryonic stem cells during their first stages of differentiation. This analysis may be useful in directed differentiation of embryonic stem cells for generating therapeutically viable cells. [unreadable] [unreadable] [unreadable]